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The Journal of Immunology, 2007, 178: 1301-1311.
Copyright © 2007 by The American Association of Immunologists, Inc.

An Alternative Pathway of NF-{kappa}B Activation Results in Maturation and T Cell Priming Activity of Dendritic Cells Overexpressing a Mutated I{kappa}B{alpha}1

Fabrice Moore*, Sofia Buonocore*, Ezra Aksoy*, Najate Ouled-Haddou*, Stanislas Goriely*, Elena Lazarova*, Frédéric Paulart*, Carlo Heirman{dagger}, Elsy Vaeremans{dagger}, Kris Thielemans{dagger}, Michel Goldman* and Véronique Flamand2,*

* Institute for Medical Immunology, Université Libre de Bruxelles, Gosselies, Belgium; and {dagger} Laboratory of Physiology, Medical School of Vrije Universiteit, Brussels, Belgium


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Maturation of dendritic cells (DC) is a critical step in the induction of T cell responses and depends on the activation of NF-{kappa}B transcription factors. Therefore, inhibition of NF-{kappa}B activation has been proposed as a strategy to maintain DC in an immature stage and to promote immune tolerance. Herein, we generated murine myeloid DC expressing a mutated I{kappa}B{alpha} acting as a superrepressor of the classical NF-{kappa}B pathway (s-rI{kappa}B DC) to investigate the consequences of NF-{kappa}B inhibition on the ability of DC to prime T cell responses. Upon in vitro LPS activation, maturation of s-rI{kappa}B DC was profoundly impaired as indicated by defective up-regulation of MHC class II and costimulatory molecules and reduced secretion of IL-12 p70 and TNF-{alpha}. In contrast, after injection, s-rI{kappa}B DC had the same capacity as control DC to migrate to draining lymph node and to induce Th1- and Th2-type cytokine production in a MHC class II-incompatible host mice. Likewise, s-rI{kappa}B DC pulsed with OVA were as efficient as control DC to induce Ag-specific T cell responses in vivo. Indeed, further in vitro experiments established that s-rI{kappa}B DC undergo efficient maturation upon prolonged contact with activated T cells via the alternative pathway of NF-{kappa}B activation triggered at least partly by lymphotoxin beta receptor ligation and involving processing of p100/RelB complexes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DC)3 are recognized as the most efficient APC that elicit both immunogenic and tolerogenic T cell responses and are involved in both central and peripheral tolerance (1, 2, 3, 4). One of the factors determining the effectiveness of the T cell response to Ags is the maturation status of the DC. Immature DC expressing few MHC and costimulatory molecules such as B7 and CD40 are likely to be involved in T cell unresponsiveness. In contrast, mature DC express higher amounts of MHC, costimulatory molecules, and proinflammatory cytokines secretion to efficiently prime T cell responses (5, 6, 7, 8, 9).

For years, evidence accumulated that this maturation step is highly dependent on the transcription factor NF-{kappa}B. Indeed, several reports using NF-{kappa}B decoy oligodeoxyribonucleotides (10, 11), N-acetyl-cysteine (12), or the proteasome inhibitor PSI (13) have already highlighted the major role of NF-{kappa}B in DC maturation and Ag presentation. Furthermore, loss of single (c-Rel or RelB) or multiple NF-{kappa}B members has been demonstrated to induce tolerogenic DC (14, 15) that are able to prevent experimental autoimmune encephalomyelitis development (16), to regulate diabetes in NOD mice (17), or to prolong survival of liver and cardiac allografts (10, 11, 18).

NF-{kappa}B is ubiquitously expressed and regulates a number of genes involved in cell development, inflammation, immune responses, and apoptosis (19, 20, 21). In mammalian cells, NF-{kappa}B family is composed of five proteins structurally related to each other that form homo- or heterodimers and that include NF-{kappa}B1 (p50 and its precursor p105), NF-{kappa}B2 (p52 and its precursor p100), RelA (p65), RelB, and c-Rel (22, 23). In resting cells, inactive NF-{kappa}B dimers are sequestered in the cytosol by I{kappa}B family members, which is composed of I{kappa}B{alpha}, I{kappa}Bbeta, I{kappa}B{epsilon}, Bcl-3, p105, and p100 (22, 24). Upon stimulation through TLR or cytokine receptors, I{kappa}B proteins are phosphorylated on specific serine residues by the I{kappa}B kinase (IKK) complex (composed of IKK{alpha} and IKKbeta catalytic subunits and IKK{gamma} regulatory subunit), which targets them for ubiquitination and degradation by the proteasome (25). Free NF-{kappa}B dimers are then able to translocate to the nucleus where it can bind to several gene promoters. Most receptor signaling have been demonstrated to lead to NF-{kappa}B activation through the "classical pathway," which involves phosphorylation of small I{kappa}B proteins by IKKbeta (22). However, recently, it has been shown that an "alternative pathway" of NF-{kappa}B may be induced. This pathway does not require I{kappa}B phosphorylation and degradation but depends on activation of IKK{alpha} by NF-{kappa}B-inducing kinase. The phosphorylation of IKK{alpha} leads to the processing of NF-{kappa}B2 p100 into p52 that further translocates into the nucleus in association with RelB (26, 27). In mice, IKK{alpha}-dependent alternative pathway is essential for secondary lymphoid organ development and homeostasis (28, 29, 30, 31, 32) and for nuclear translocation of p52/RelB and p50/RelB dimers downstream of lymphotoxin beta receptor (LTbetaR)-activated mouse embryonic fibroblasts (MEF) and HeLa cells (33, 34) but was never documented so far during DC maturation.

RelB is a NF-{kappa}B family member that can neither form homodimers, nor bind directly to the DNA, although it can activate gene transcription in association with p50 or p52 (35, 36). RelB has been shown to be important for the function of Ag-presenting DC populations of the lymphoid tissues, and studies on RelB-deficient (–/–) mice highlight its role in lymphoid organs development and homeostasis (37, 38, 39). Moreover, RelB regulation has been shown to differ consistently from other NF-{kappa}B family members, as it was demonstrated to bind only weakly I{kappa}B{alpha} (40, 41), but to be mostly associated with NF-{kappa}B2 p100 in the cytosol (33), which places RelB as a major component of NF-{kappa}B dimers activated by the alternative pathway.

In this study, we used a retrovirus-based method to express a mutated I{kappa}B{alpha} inhibitory protein (superrepressor of the classical NF-{kappa}B pathway (s-rI{kappa}B) in which Ser32 and Ser36 are replaced by alanine) that cannot be degraded and that acts therefore as a superrepressor of NF-{kappa}B activity in bone marrow-derived DC (BM-DC). We tested the ability of the modified DC to undergo maturation and to activate T cell responses in vitro and in vivo. We demonstrated that, although s-rI{kappa}B DC were profoundly inhibited to mature in response to LPS due to inhibition of the classical NF-{kappa}B pathway, they remained fully competent to mature when came in contact with T cells. In accordance with this finding, we show evidence that in s-rI{kappa}B DC an alternative pathway of NF-{kappa}B is activated upon T cell contact similar to the alternative NF-{kappa}B pathway mediated by the engagement of their surface LTbetaR.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Eight- to 12-wk-old C57BL/6.C-H-2-bm12 (bm12) mice, which differ from C57BL/6 (H-2b) mice with three point mutations in the I-Abeta-chain, were obtained from The Jackson Laboratory. Sex-matched C57BL/6 (H-2b) and BALB/c (H-2d) mice were purchased from Harlan Netherlands. DO11.10 TCR-transgenic mice (42) were provided by M. Moser (IBMM, Université Libre de Bruxelles, Gosselies, Belgium). All animals were used at 6–10 wk of age and bred in our specific pathogen-free animal facility.

Abs, reagents, and cell lines

For DC activation, LPS from Escherichia coli (serotype 0111:B4; Sigma-Aldrich) was added at 100 ng/ml; polyinosine-polycytidylic acid (poly(I:C)) and (S-[2.3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-Lys4-OH) trihydrochloride (Pam3CSK4) were purchased from GE Healthcare and used at 10 and 1 µg/ml, respectively; anti-mouse LTbetaR mAb (5G11b; Serotec) was used at 10 µg/ml for DC activations and revealed with FITC-conjugated F(ab')2 mouse anti-rat IgG (Jackson ImmunoResearch Laboratories) for cytometry analysis. CFSE was purchased from Molecular Probes. Chicken OVA peptide (OVA 323–339) was obtained from NeoMPS. The PhoenixECO ecotropic packaging cell line were grown in DMEM containing 4.5 g/L glucose and L-glutamine supplemented with 5% heat-inactivated FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin (BioWhittaker).

Generation and phenotyping of BM-DC

DC were propagated from bone marrow progenitors in GM-CSF-containing medium and harvested after 7 days as described previously (43). Briefly, bone marrow was flushed from the femurs and the tibiae of mice, disintegrated by vigorous pipetting, filtered through a nylon mesh, and depleted of RBC with ammonium chloride. At day 0, bone marrow progenitors were seeded in a 6-well plate at the rate of 106 cells/well in 4 ml of 20 ng/ml recombinant murine GM-CSF containing RPMI 1640 (BioWhittaker)/10% heat-inactivated FBS (SB0012; BioWhittaker)/20 mM HEPES/2 mM glutamine, 1 mM nonessential amino acids (BioWhittaker)/sodium pyruvate (BioWhittaker)/2-ME and were fed at days 3 and 6 with fresh medium. For phenotypical analysis, cells were suspended in PBS/0.5% (w/v) BSA supplemented with 0.1% (w/v) sodium azide. Nonspecific binding of mAb to FcR was blocked by preincubating cells with culture supernatant of anti-Fc{gamma}RII/III mAb (2.4G2). The following reagents and mAb were obtained from BD Pharmingen: biotinylated mAbs directed against CD80 (1G10), CD86 (GL1), or CD40 (3/23) surface molecules, PE-labeled streptavidin, FITC-conjugated anti-CD11c mAb (HL3), and unlabeled anti-I-Ab,d,q/I-Ed,k mAb (M5/114.15.2). FITC-conjugated F(ab')2 mouse anti-rat IgG was purchased from Jackson ImmunoResearch Laboratories. Cellular viability was assessed using Annexin VFITC/propidium iodide double staining as described previously (44). Briefly, cells were collected, washed with PBS, and resuspended at 106 cells/ml in 1x annexin V-binding buffer (BD Pharmingen) with 1 µg/ml Annexin VFITC (BD Pharmingen) and 1 µM propidium iodide (Sigma-Aldrich). After 15 min, labeling was stopped by adding 5 volumes of binding buffer and cells were analyzed by flow cytometry within the hour on a Cyan ADP flow cytometer (DakoCytomation).

Cloning of retroviral vector constructs

For retrovirus production, the retroviral vector MFG derived from Moloney murine leukemia virus was used. pMFGP1A retroviral vector (43) was used as control. pMFGeGFP was used to assess transduction efficiency. The enhanced GFP (eGFP) gene was obtained as a NcoI-BclI fragment by digestion of peGFP-C1 (Clontech Westburg) and ligated in pMFG/NcoI-BamHI. The pMFGs-rI{kappa}B vector was constructed as followed: the s-rI{kappa}B gene was amplified from the pRCs-rI{kappa}B with the forward primer 5'-TTTggCCTCATgAgTTACCCATACgATgT-TCCA-3' (containing a BspHI restriction site underlined) and the reverse primer 5'-CCCAgATCTTCATAACgTCAgACgCTgg-3' (containing a BglII restriction site underlined). After cloning in pCR-BluntII-TOPO (Invitrogen-Merelbeke), the s-rI{kappa}B gene was excised from the plasmid as a BspHI-BglII fragment and cloned in pMFG/NcoI-BamHI. In DC, expression of P1A (control protein), eGFP, and s-rI{kappa}B proteins were all under the control of the retroviral MFG promoter (5' LTR from Moloney murine leukemia virus).

Retrovirus production and DC transduction

Ten million PhoenixECO producer cells were transfected with 40 µg of retroviral vector DNA by the calcium phosphate precipitation method (45). Cells were incubated in complete DMEM supplemented with 25 µM chloroquine (Sigma-Aldrich) at 37°C for 10 h. The medium was then changed with complete DMEM and renewed after another 14 h with Opti-MEM (Invitrogen Life Technologies). Retrovirus-containing medium was harvested 48 h after transfection. The retroviral supernatants were filtered (0.45-µm pore size), snap-frozen, and stored at –80°C. On days 1, 2, and 3 after the start of the bone marrow cell culture, the medium was removed and replaced with 2 ml of viral supernatant containing 8 µg/ml polybrene (Sigma-Aldrich). The cells were transduced by centrifugation of the 6-well plates for 2 h at 2400 rpm and at room temperature. The retroviral supernatant was removed, and the cells were suspended in GM-CSF-containing medium.

RNA purification and RT-PCR for TLR mRNA levels evaluation

Messenger RNA was extracted from 106 control-transduced DC (Ctrl DC) or s-rI{kappa}B DC using a MagnaPure LC mRNA Isolation Kit I (Roche Diagnostics). Purified mRNA was next converted to cDNA using ImProm II Reverse Transcription System (Promega). The resulting cDNA was purified using Wizard DNA Clean Up System (Promega), resuspended in 40 µl of nuclease-free water and cDNA concentration was evaluated by spectrophotometry at OD260 nm. One hundred nanograms was then amplified by PCR for a nonsaturating number of cycles with the following primers: mouse TLR2 sense 5'-CAGCTTAAAGGGCGGGTCAGAG-3', and mouse TLR2 antisense, 5'-TGGAGAGACGCCAGCTCTGGCTCA-3'; mouse TLR3 sense, 5'-GCTCATTCTCCCTTGCTCAC-3', and mouse TLR3 antisense, 5'-CCCGAAAACATCCTTCTCAA-3'; and mouse TLR4 sense, 5'-TATTCCCTCAGCACTCTTGATT-3', and mouse TLR4 antisense, 5'-GTAGTGAAGGCAGAGGTGAAAG-3'. As input control, we used primers for mouse beta-actin (sense, 5'-GGACTCCTATGTGGGTGACGAGG-3', and antisense, 5'-GGGAGAGCATAGCCCTCGTAGAT-3'). Amplification products were resolved on a 1% agarose gel containing SYBR Safe DNA gel stain (Invitrogen Life Technologies) and visualized with a Chemidoc EQ (Bio-Rad).

Proliferation assays and cytokines production in MLC

For proliferation assays, 105 C57BL/6 T cells from draining popliteal and inguinal lymph nodes (LN) of naive or primed (14 days after the immunization) mice were cocultured for 72 h in 96-well round-bottom plates with an increasing numbers of bm12 DC (primary MLC) or gamma-irradiated (20 Gy) bm12 splenocytes (secondary MLC) as stimulators. A total of 1 µCi/well [3H]methylthymidine (ICN Biomedicals) was added for the final 16 h. [3H]Thymidine incorporation was measured by liquid scintillation counting. Results are expressed as mean cpm. For cytokine production assays, 106 C57BL/6 cells from draining popliteal and inguinal LN of primed mice were cocultured in a 48-well plate with either 2.5 x 106 gamma-irradiated splenocytes from either syngeneic (C57BL/6 mice), allogeneic (bm12 mice), or third-party origin (BALB/c mice). Supernatants were harvested after 24 h of culture for determination of IL-2 levels and after 72 h for all the other cytokines. Quantification of cytokines in MLC supernatants was made using commercially available ELISA:Duoset (R&D Systems) for IFN-{gamma}, IL-2, IL-4, and IL-13; Opt EIA set (BD Pharmingen) for IL-5 and CytoSet (BioSource International) for IL-10.

CD4+ T cell isolation and in vivo T cell proliferation

Total LN cells from DO11.10 TCR-transgenic mice were incubated with FITC-conjugated anti-CD4 Abs in 500 µl of cold PBS supplemented with 0.5 mM EDTA and 0.5% BSA (20 min at 4°C). Cells were washed with cold PBS and incubated another 20 min with anti-FITC mAb-coupled microbeads (Miltenyi Biotec) in cold PBS/0.5 mM EDTA/0.5% BSA. CD4+ T cells were further enriched to >95% purity by positive selection over a MACS separation column (Miltenyi Biotec) and labeled with CFSE. A total of 106 CFSE-labeled CD4+ T cells isolated from DO11.10 TCR-transgenic mice was transferred into BALB/c-recipient mice by i.p. injection. Two days after reconstitution, recipient mice were immunized s.c. either with 3 x 105 BALB/c-unpulsed or OVA-pulsed Ctrl or s-rI{kappa}B DC. Proliferation of DO11.10-transgenic T cells was evaluated 3 days after immunization with a flow cytometer by CFSE intensity decrease.

Migration Assay by 111In oxine labeling and biodistribution analysis

Ctrl or s-rI{kappa}B DC were suspended in 0.1 ml of complete culture medium and incubated for 30 min at room temperature with 200 µCi of 111In-oxine (Tyco Healthcare). PBS washed cells were then footpad injected at 1.106 DC/50 µl. A negative control mouse was injected with 50 µCi of pure 111In oxine in 50 µl of PBS. Eighteen to 20 h after DC administration, mice were sacrificed and migration to the draining LN was imaged with a gamma camera (SMV DSX; Sopha). Results are expressed as the percentage of the whole body radioactivity that could be measured in the popliteal LN after background correction.

Isolation of DC from MLC

Total cells were harvested from MLC and washed with cold PBS. From that step, cells were always kept at 4°C to avoid unintended activation. Cells were then separated by density centrifugation using a Nycodenz gradient (density = 1.085 g/ml; Nycomed) at 1700 x g (15 min/4°C). Low-density floating cells were then incubated with anti-mouse CD11c (N418) mAb-loaded microbeads (Miltenyi Biotec) in 100 µl of cold PBS supplemented with 0.5 mM EDTA and 0.5% BSA (20 min at 4°C). CD11c+ DC were further purified to >95% purity by positive selection over a MACS separation column (Miltenyi Biotec).

Immunoblotting

BM-DC were harvested, washed twice with cold PBS and directly lysed in cell lysis reagent (10 mM Tris-Hcl, 150 mM NaCl, 1.5 mM MgCl2, and 1% Igepal). Protein concentration was evaluated by Bradford assay (Bio-Rad). An equal amount of proteins was resolved by 8–10% SDS-PAGE and immunoblotted. Membranes were then probed with the following Abs: anti-I{kappa}B{alpha} (sc-371), anti-RelA (sc-109), anti-RelB (sc-226), anti-c-Rel (sc-6955), anti-NF-{kappa}B1 (sc-1190), anti-NF-{kappa}B2 (sc-7386), anti-USF-2 (sc-871), anti-IKK{alpha} (sc-7182), anti-IKKbeta (sc-8014) (Santa Cruz Biotechnology), anti-phospho-IKK{alpha}/IKKbeta (2681; Cell Signaling Technology), and anti-GAPDH (H86504M; Biodesign International). Immunoreactive bands were revealed using the ECL detection method (Amersham Biosciences). For coimmunoprecipitation analysis, whole cell extracts (WCE) of Ctrl or s-rI{kappa}B DC were lysed in 250 µl of buffer containing (50 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Igepal, and protease inhibitors (Roche; 1 tablet/50 ml)) and then incubated overnight at 4°C under agitation with 2 µg of anti-RelB Ab. WCE were then incubated with a 1:1 slurry of protein A:protein G-Sepharose beads (Amersham Biosciences) for 2 h at 4°C. The beads were then washed five times with lysis buffer and suspended in denaturing sample buffer. Coimmunoprecipitated complexes were then resolved by 8% SDS-PAGE and immunoblotted. Membranes were then probed with anti-NF-{kappa}B2 (sc-7386) and anti-RelB (sc-226) Abs. Mouse TrueBlot HRP-conjugated anti-mouse IgG (eBioscience) was used as secondary Ab for NF-{kappa}B2 revelation, to avoid IgG interference in the p52 bands. Immunoreactive bands were revealed using the ECL detection method (Amersham Biosciences).

EMSA

Nuclear extracts from BM-DC were prepared as described previously (46). The sequence of NF-{kappa}B consensus probe was as follows: 5'-AGTTGAGGGACTTTCCCAGGC-3' (Proligo) and the sequence of the NF-{kappa}B mutated probe used for competition assay was 5'-AGTTGAGGCGACTTTCCCAGGC-3'. The NF-{kappa}B consensus oligonucleotide from the upstream regulatory element of the c-myc gene was 5'-GATCCAAGTCCGGGTTTTCCCCAACC-3' (47) (Proligo). Core elements are underlined. The EMSA for NF-{kappa}B was conducted as reported previously (48). For supershift assays, mAb against p52, p50, RelA, RelB or c-Rel (described above) were added to the binding reaction at a final concentration of 2 µg/reaction mixture. As loading control, the same nuclear extracts were tested for binding to the Sp1 consensus probe: 5'-GTGGTGGGCGGGGTGTCCCGCCCGCCTG-3'.

Statistical analysis

Statistical analysis was performed using the two-tailed Mann-Whitney nonparametric test (p < 0.05 was considered significant).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Reduced NF-{kappa}B DNA-binding activity in s-rI{kappa}B-transduced DC

C57BL/6bm12 (bm12) DC were propagated from bone marrow progenitors in GM-CSF-containing medium. Transductions with either control (MFGP1A) or s-rI{kappa}B-encoding retroviruses (MFGs-rI{kappa}B) were performed on days 1, 2, and 3, and the cells were cultured in GM-CSF-containing medium until day 7. Using a parallel transduction with a retrovirus encoding eGFP, we established by flow cytometry that the average transduction efficiency at the end of the culture was ~80% (data not shown). On day 7, cells were collected, and the accurate expression and functionality of the s-rI{kappa}B was assessed. First, by immunoblotting assays, we observed that DC transduced with MFGs-rI{kappa}B (s-rI{kappa}B DC) highly expressed the superrepressor I{kappa}B compared with Ctrl DC. s-rI{kappa}B carries a triple hemagglutinin tag that distinguishes it from endogenous I{kappa}B{alpha} (Fig. 1A). We then performed EMSA to test whether the expression of the s-rI{kappa}B protein would affect NF-{kappa}B DNA-binding activity in response to a stimulus such as bacterial LPS. Analysis of EMSA performed using a NF-{kappa}B consensus probe revealed that NF-{kappa}B DNA-binding activity was drastically increased in Ctrl DC stimulated with LPS for 1 h compared with their unstimulated counterparts and that the NF-{kappa}B DNA-binding activity decreased after 24 h of LPS stimulation (Fig. 1B). Specificity of the shifted bands was confirmed by competition assays with consensus and mutated cold probes (Fig. 1B). Furthermore, supershift experiments allowed us to identify the NF-{kappa}B-binding complex as the classical p50/p65 dimer (Fig. 1C). As shown in Fig. 1B, LPS-induced p50/p65 DNA-binding activity was strongly repressed in s-rI{kappa}B DC at both timings of LPS stimulation. Immunoblot analysis of nuclear extracts from LPS-activated Ctrl or s-rI{kappa}B DC also confirmed the inhibition of p65 nuclear translocation in s-rI{kappa}B DC in response to 1 h of stimulation with LPS (Fig. 1D) that was neither compensated by any of the other NF-{kappa}B subunits, nor restored after 24 h of LPS exposure (Fig. 1D). Those results confirmed the expression and the repressor activity of the s-rI{kappa}B in bm12 BM-DC. We next tested Ctrl or s-rI{kappa}B DC viability by performing annexinV-propidium iodide double staining. Ctrl DC that were frozen and thawed twice were used as positive control for dead cells. As shown in Fig. 1E, the viability of s-I{kappa}B DC was not affected compared with Ctrl DC.


Figure 1
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  		FIGURE 1. s-rI{kappa}B is expressed in MFGs-rI{kappa}B-transduced DC and inhibits NF-{kappa}B nuclear translocation. DC were propagated from bm12 bone marrow progenitors in GM-CSF-containing medium. Transductions with control (Ctrl DC) or MFGs-rI{kappa}B retroviruses (s-rI{kappa}B DC) were performed on days 1, 2, 3, and cells were cultured in GM-CSF-containing medium until day 7. A, At the end of the culture (day7), total cell lysates from Ctrl or s-rI{kappa}B DC were prepared, and 10 µg of proteins was resolved by 10% SDS-PAGE, immunoblotted, and then probed with anti-I{kappa}B{alpha} Abs. One representative experiment is shown. B, On day 7, nuclear extracts were prepared from Ctrl or s-rI{kappa}B DC that were left untreated or stimulated with LPS for 1 or 24 h. Proteins (5 µg) were subjected to EMSA using a NF-{kappa}B consensus probe or a Sp1 probe. For competition assay, decreasing concentrations (80- to 5-fold molar excess) of consensus or mutated competitor cold probe were added. C, Nuclear extracts (5 µg) from LPS-stimulated Ctrl DC were subjected to supershift EMSA using a NF-{kappa}B consensus probe and either anti-p50, anti-p65, anti-p52, anti-RelB, or anti-c-Rel Abs. D, Nuclear proteins (20 µg) of were resolved by 8% SDS-PAGE, immunoblotted, and probed with anti-RelB, anti-NF-{kappa}B2 (p100/p52), anti-NF-{kappa}B1 (p105/p50), anti-RelA (p65), anti-c-Rel, and anti-USF-2 Abs. E, Ctrl or s-rI{kappa}B DC were collected, washed with PBS, and incubated in annexin V binding buffer containing 1 µg/ml Annexin VFITC and 1 µM propidium iodide. Ctrl DC that were frozen and thawed twice were used as positive control for dead cells. After 15 min, labeling was stopped, and cells were analyzed by flow cytometry within the hour.

 
s-rI{kappa}B DC are unable to mature in response to TLR ligands

Since NF-{kappa}B has been described as a major factor in the DC maturation processes (10, 11, 12, 49), we investigated whether the expression of the s-rI{kappa}B protein would affect DC ability to mature in response to different types of stimuli. To this end, Ctrl or s-rI{kappa}B DC were exposed for 24 h to LPS (a TLR4 agonist), poly(I:C) (a TLR3 agonist), or Pam3CSK4 (a TLR2 agonist) and analyzed by flow cytometry for the surface expression of MHC and costimulatory molecules. The expression of MHC class II (MHC II), CD80, CD86, and CD40 was significantly increased at the surface of Ctrl DC in response to each type of stimulus, while CD11c expression slightly decreased. In contrast, untreated s-rI{kappa}B DC displayed a reduced basal expression of MHC II, CD80, CD86, and CD40 molecules compared with untreated Ctrl DC and were unable to significantly up-regulate any of those markers upon stimulation (Fig. 2A). The inhibition of maturation observed for s-rI{kappa}B DC in response to LPS, poly(I:C), or Pam3CSK4 was not due to a lack of corresponding TLR expression because TLR4, TLR3, and TLR2 mRNA levels were equal in Ctrl and s-rI{kappa}B DC (Fig. 2B). Interestingly, we marked that the CD11c expression was strongly reduced in s-rI{kappa}B DC, suggesting that DC differentiation might be partially affected in these cells.


Figure 2
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  		FIGURE 2. s-rI{kappa}B DC are unable to mature in response to TLR ligands. A, Ctrl or s-rI{kappa}B BM-DC were generated in 7 days and then left untreated or exposed either to 100 ng/ml LPS, 10 µg/ml poly(I:C), or 1 µg/ml Pam3CSK4. After 24 h, cells were analyzed by flow cytometry for the surface expression of MHC and costimulatory molecules. Mean fluorescence intensity (MFI) ± SEM are represented (*, p < 0.05, considered as significant). B, mRNA was extracted from 106 Ctrl or s-rI{kappa}B DC and converted to cDNA by reverse transcription. One hundred nanograms of cDNA from Ctrl or s-rI{kappa}B DC was then amplified by PCR for a nonsaturating number of cycles with primers for mouse TLR2, TLR3, TLR4, and beta-actin. Amplification products were then resolved on a 1% agarose gel. C, Ctrl or s-rI{kappa}B DC were stimulated or not with LPS (100 ng/ml) for the last 24 h of the culture. Supernatants were harvested, and the concentration of IL-12 p70 and TNF-{alpha} were evaluated by ELISA. Mean cytokine production/ml/106 cells ± SD are represented (*, p < 0.05; considered as significant).

 
We then focused on LPS signaling, since TLR-4 triggering activates NF-{kappa}B through MyD88-dependent and MyD88-independent manner (50, 51). We assessed by ELISA the ability of s-rI{kappa}B DC to produce IL-12 p70 and TNF-{alpha} in response to LPS. As shown in Fig. 2C, IL-12 p70 levels were low for unstimulated Ctrl and s-rI{kappa}B DC. Still, while IL-12 p70 secretion was markedly increased in LPS-stimulated Ctrl DC, LPS-stimulated s-rI{kappa}B DC showed impaired production of this cytokine. LPS-induced TNF-{alpha} production was also significantly decreased in s-rI{kappa}B DC, however, to a lesser degree. Taken together, these results indicated a broad defect in s-rI{kappa}B DC maturation capacity in response to several TLR ligands tested.

s-rI{kappa}B DC conserve migratory capacities and stimulate in vivo CD4+ T cells proliferation

During maturation, DC undergo modifications in their chemotactic properties (52, 53, 54). The MIP-1{alpha} receptor (i.e., CCR5) expressed on immature DC is down-regulated upon DC maturation. Mature DC express MIP-3beta receptor (i.e., CCR7), which allows them to migrate to the secondary lymphoid organs. As NF-{kappa}B has been described as an important regulator of CCR7 expression (55, 56), we first wanted to test whether s-rI{kappa}B DC had the capacity to migrate in vivo. We first observed by quantitative RT-PCR analysis that s-rI{kappa}B DC down-regulated CCR5 mRNA and up-regulated CCR7 mRNA upon LPS stimulation in a similar manner as Ctrl DC (data not shown). Next, we performed migration assays based on gamma ray emission with 111In oxine-labeled bm12 Ctrl or s-rI{kappa}B DC inoculated in footpads of MHC II-incompatible C57BL/6-recipient mice. As shown in Fig. 3A, Ctrl or s-rI{kappa}B DC migrated equally to the draining popliteal LN.


Figure 3
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  		FIGURE 3. s-rI{kappa}B DC conserve migratory capacities and stimulate in vivo CD4+ T cells proliferation. A, A total of 1 x 106 111In oxine-labeled Ctrl or s-rI{kappa}B BM-DC from bm12 mice was footpad injected in MHC II-incompatible C57BL/6 wild-type recipient mice. After 18 h, mice were sacrificed, and gamma ray emission of the draining popliteal LN was measured with a gamma camera (SMV DSX; Sopha). Results are expressed as the percentage of the whole body radioactivity that could be measured in the popliteal LN after background correction. B, CFSE-labeled CD4+ T cells isolated from DO11.10 TCR-transgenic mice were transferred into BALB/c-recipient mice by i.p. injection. Two days after reconstitution, recipient mice were immunized s.c. with either 3 x 105 BALB/c unpulsed Ctrl DC, unpulsed s-rI{kappa}B DC, OVA-pulsed Ctrl DC, or OVA-pulsed s-rI{kappa}B DC. Proliferation of DO11.10-transgenic T cells was evaluated 3 days after immunization with a flow cytometer by CFSE intensity decrease. The results are expressed as the mean percentage of DO11.10-transgenic T cells that underwent 0–6 divisions ± SD (*, p < 0.05, considered as significant).

 
Finally, we evaluated the tolerogenic properties of those s-rI{kappa}B DC by testing their ability to prime T cell proliferation in vivo. For that purpose, we transferred CFSE-labeled OVA-specific TCR-transgenic (DO11.10) CD4+ T cells into syngeneic BALB/c recipients. Mice were then immunized with either unpulsed or OVA-pulsed Ctrl DC or s-rI{kappa}B DC. Proliferation of DO11.10-transgenic T cells was evaluated 3 days after immunization by flow cytometry. As expected, no T cell proliferation was elicited by unpulsed Ctrl or s-rI{kappa}B DC (Fig. 3B). Surprisingly, we observed that OVA-pulsed s-rI{kappa}B DC primed transgenic T cell proliferation as efficiently as OVA-pulsed Ctrl DC in terms of either percentage of proliferating cells or number of cell divisions.

s-rI{kappa}B DC efficiently prime immune response against MHC II alloantigens

In the next set of experiments, we aimed to study whether allogeneic s-rI{kappa}B DC would inhibit T cell responses in vivo. C57BL/6-recipient mice were submitted to repeated i.v. injections of Ctrl or s-rI{kappa}B bm12 DC. Two weeks after the final injection, MLC were prepared with LN cells and donor type splenocytes as stimulators for the proliferation assay. LN cells that were collected from mice injected with either allogeneic Ctrl or s-rI{kappa}B DC were equally primed to proliferate in response to the donor alloantigens (data not shown). MLC were then performed with the cells of the draining LN from immunized C57BL/6 mice and syngeneic (C57BL/6), donor-type (bm12), or third-party (BALB/c) splenocytes as stimulators for cytokine measurement by ELISA. As shown in Fig. 4, bm12 Ctrl or s-rI{kappa}B DC induced similar levels of IL-10 and Th1- or Th2-type (IFN-{gamma}, IL-4, IL-5, and IL-13) cytokines in C57BL/6 responder cells. In accordance with the proliferation assays, similar level of IL-2 was produced in response to alloantigens in mice treated with Ctrl or s-rI{kappa}B DC. Similar results were obtained after a single injection of Ctrl or s-rI{kappa}B DC (data not shown). These data indicated that, even though s-rI{kappa}B DC display an altered immature phenotype in vitro, they remain fully competent to prime a T cell response in vivo.


Figure 4
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  		FIGURE 4. Immunization with allogeneic s-rI{kappa}B DC does not inhibit T cell cytokine production. Six- to 8-wk-old C57BL/6 mice were i.v. injected three times, once a week, with 2 x 106 bm12 Ctrl- or s-rI{kappa}B BM-DC. Noninjected C57BL/6 mice were used as controls. Seven days after the last injection, 106 LN cells were cocultured with 2.5 x 106 gamma-irradiated syngeneic (C57BL/6), allogeneic (bm12), or third-party (BALB/) splenocytes. Culture supernatants were harvested after 24 h for IL-2 measurement and after 72 h for all the other cytokines detection by ELISA. Mean cytokine production/ml ± SD are represented (*, p < 0.05 considered as significant).

 
Allogeneic s-rI{kappa}B DC are able to undergo maturation and encompass NF-{kappa}B activation in contact with T cells

To determine how s-rI{kappa}B DC that are unable to respond to TLR ligands still are able to prime T cell responses in vivo, we tested whether s-rI{kappa}B DC could undergo T cell-dependent maturation. We used an in vitro approach that allowed us to monitor s-rI{kappa}B DC phenotype during T cell contact. Hence, MLC were prepared with CFSE-labeled bm12 Ctrl or s-rI{kappa}B DC and C57BL/6-purified CD4+ T cells. CFSE-positive DC were analyzed by flow cytometry at 72 h. As shown in Fig. 5A, Ctrl DC up-regulated the MHC II and CD80 molecules after T cell contact. CD11c surface expression was slightly decreased in those DC. Unexpectedly, CD11c expression was strongly up-regulated in s-rI{kappa}B DC after T cell encounter. After 72 h of MLC, the phenotype of s-rI{kappa}B DC was analogous to Ctrl DC in terms of MHC II and CD80 up-regulation. Moreover, we observed that purified C57BL/6 CD4+ T cells were equally primed to proliferate in response to Ctrl or s-rI{kappa}B bm12 DC in this primary MLC (Fig. 5B).


Figure 5
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  		FIGURE 5. Allogeneic s-rI{kappa}B DC are able to undergo maturation and encompass NF-{kappa}B activation in contact with T cells. A, MLC were prepared with CFSE-labeled bm12 Ctrl or s-rI{kappa}B DC and C57BL/6-purified CD4+ T cells. Total cells were collected from MLC at 72 h and were analyzed by flow cytometry for the expression of CD11c, MHC II, and CD80, gating on CFSE-positive cells. B, A total of 105 C57BL/6 T cells of naive mice were cocultured for 72 h in 96-well round-bottom plates with an increasing numbers of bm12 Ctrl or s-rI{kappa}B DC as stimulators. A total of 1 µCi/well [3H]methylthymidine was added for the final 16 h. [3H]Thymidine incorporation was measured by liquid scintillation counting. Results are expressed as mean cpm ± SD. C, Ctrl and s-rI{kappa}B DC were isolated from MLC using a density gradient followed by a magnetic sorting with anti-CD11c mAb-coupled microbeads. Isolation was performed at 4°C to avoid unintended activation. DC purity was assessed by CFSE-positive cells measurement. D, Nuclear extracts were prepared from unstimulated or 48 h of MLC-stimulated Ctrl or s-rI{kappa}B DC, and 5 µg of proteins was subjected to EMSA using a NF-{kappa}B c-myc probe. E, Ten micrograms of nuclear proteins was resolved by 8% SDS-PAGE, immunoblotted, and probed with anti-RelB, anti-NF-{kappa}B2 (p100/p52), anti-NF-{kappa}B1 (p105/p50), anti-RelA (p65), anti-c-Rel, and anti-USF-2 Abs.

 
We then hypothesized that: 1) NF-{kappa}B activity is restored in s-rI{kappa}B DC during T cell contact and 2) NF-{kappa}B activity occurs in s-rI{kappa}B DC through the I{kappa}B-independent alternative pathway that implicates nuclear migration of p52/RelB dimers. To investigate both queries, MLC were prepared between CFSE-labeled bm12 Ctrl or s-rI{kappa}B DC and C57BL/6-purified CD4+ T cells. After 48 h, bm12 Ctrl or s-rI{kappa}B DC were isolated from MLC with anti-CD11c mAb-loaded microbeads. Isolation was performed at 4°C to avoid unintended activation. DC purity assessed by CFSE-positive cells measurement reached 95% (Fig. 5C). Nuclear extracts were prepared, and EMSA were performed using the NF-{kappa}B-binding sequence from the upstream regulatory element of the c-myc gene as probe that had already been shown to bind p52/RelB dimers efficiently (57). Few NF-{kappa}B-binding complexes were detected in both types of resting DC (Fig. 5D). However, this NF-{kappa}B-binding activity was drastically increased in allogeneic Ctrl and s-rI{kappa}B DC after 48 h of CD4+ T cell contact. Furthermore, we established by immunoblotting that the NF-{kappa}B-binding complexes contained both RelB and NF-{kappa}B2 p52 (Fig. 5E) while no RelA (p65) or c-Rel could be detected (Fig. 5E). NF-{kappa}B1 p50 levels remained rather stable, and we cannot exclude its participation in the activated NF-{kappa}B dimers (Fig. 5E). Equal loading and absence of cytoplasmic contamination were verified respectively by USF-2 (Fig. 5E) and GAPDH probing (data not shown).

The alternative NF-{kappa}B pathway can be induced in s-rI{kappa}B DC through LTbetaR engagement

To dissect the activation of NF-{kappa}B through the alternative pathway in s-rI{kappa}B DC, we mimicked T cell contact by engaging LTbetaR on these cells. Indeed, both the LTbetaR ligands LT{alpha}1beta2 and its homolog LIGHT have been shown to be expressed on activated murine T cells (58), and the engagement of LTbetaR was already reported to induce NF-{kappa}B activity through both the classical and the alternative pathway (33, 59). Hence, we tested if LT-LTbetaR interaction had a role in T cell-driven activation of NF-{kappa}B through the alternative pathway in s-rI{kappa}B DC. We first measured by flow cytometry that LTbetaR was equally expressed on Ctrl and s-rI{kappa}B DC (data not shown). Ctrl or s-rI{kappa}B DC were either left untreated or exposed to LPS for 1 h or to anti-LTbetaR agonist mAb for 8 h, which we and others (33, 59) have observed as the best timing for the alternative activation of NF-{kappa}B through LTbetaR. Cytoplasmic and nuclear extracts were prepared and analyzed by immunoblotting of cytoplasmic (Fig. 6A) or nuclear fractions (Fig. 6B). Cytoplasmic NF-{kappa}B2 p52 but not p100 was detected in untreated Ctrl DC and was reduced after activation of LTbetaR. In contrast, only NF-{kappa}B2 p100 was found in the cytoplasm of untreated s-rI{kappa}B DC that also decreased after LTbetaR engagement (Fig. 6A). NF-{kappa}B1 p105 expression was constant and unaffected by both types of stimuli (data not shown). Strikingly, RelB could only be detected in the cytoplasm of LTbetaR-stimulated Ctrl DC, whereas a large cytoplasmic pool of RelB was present in untreated and LPS-stimulated s-rI{kappa}B DC, and it slightly diminished after anti-LTbetaR treatment (Fig. 6A). Even more interesting, while both phosphorylation of IKK{alpha} and IKKbeta was induced in LPS- and anti-LTbetaR-treated Ctrl DC, only phospho-IKK{alpha} could be observed in s-rI{kappa}B DC, which was correlated with a slight increase of total IKK{alpha} in s-rI{kappa}B DC (Fig. 6A). Analysis of the nuclear fractions corroborated the observations made with cytoplasmic fractions. Indeed, we observed an increase of nuclear p52 and RelB levels in anti-LTbetaR mAb-treated Ctrl and s-rI{kappa}B DC (Fig. 6B). Finally, p65 nuclear translocation was only observed in LPS-treated Ctrl DC while mostly inhibited for LPS-stimulated s-rI{kappa}B DC (Fig. 6B). Taken together, these results highlight the fact that LTbetaR stimulation in s-rI{kappa}B DC strongly engages the alternative pathway of NF-{kappa}B activation involving activation of IKK{alpha} and nuclear translocation of RelB and NF-{kappa}B2 p52.


Figure 6
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  		FIGURE 6. The alternative pathway of NF-{kappa}B activation can be engaged in s-rI{kappa}B DC through LTbetaR ligation. A, Cytoplasmic and nuclear extracts were prepared from Ctrl or s-rI{kappa}B BM-DC either unstimulated, stimulated with LPS for 1 h, or stimulated with anti-LTbetaR mAb-for 8 h. Cytoplasmic extracts (20 µg) were resolved by 8% SDS-PAGE, immunoblotted, and probed with an anti-NF-{kappa}B2 (p100/p52), anti-RelB, anti-phospho-IKK{alpha}/IKKbeta, anti-IKK{alpha}, anti-IKKbeta, and anti-GAPDH Abs. B, Nuclear extracts (20 µg) from unstimulated, LPS-stimulated, and anti-LTbetaR mAb-stimulated Ctrl or s-rI{kappa}B DC were resolved by 8% SDS-PAGE, immunoblotted, and probed with anti-NF-{kappa}B2 (p100/p52), anti-RelB, anti-RelA (p65), and anti-USF-2 Abs.

 
NF-{kappa}B2 p100 processing allows RelB nuclear translocation in s-rI{kappa}B DC

Although strongly suggested by the results obtained for cytoplasmic and nuclear fractions, proof remained to be made that RelB associates with NF-{kappa}B2 p100 in unstimulated s-rI{kappa}B DC. To this end, WCE of unstimulated and LTbetaR-stimulated Ctrl and s-rI{kappa}B DC were immunoprecipitated with anti-RelB Ab and analyzed for the presence of coimmunoprecipitated NF-{kappa}B2 p100 (Fig. 7). NF-{kappa}B2 p100 was only detected in unstimulated s-rI{kappa}B DC and was partly processed to p52 after LTbetaR engagement. Immunoblotting analysis confirmed the increased amounts of RelB protein in s-rI{kappa}B DC. NF-{kappa}B2 and RelB could not be detected from the whole cell lysates of Ctrl DC, probably because not many p100 is expressed and only a few p52 interacted physically with RelB. These data confirmed that nuclear translocation of p52/RelB dimers in s-rI{kappa}B DC is associated with processing of the cytoplasmic NF-{kappa}B2 p100/RelB complexes.


Figure 7
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  		FIGURE 7. NF-{kappa}B2 p100 processing allows RelB nuclear translocation in s-rI{kappa}B DC. WCE from 3 x 106 unstimulated and anti-LTbetaR mAb-stimulated Ctrl and s-rI{kappa}B DC were immunoprecipitated with anti-RelB Ab coupled with protein A:protein G-Sepharose beads (1:1) at 4°C. Coimmunoprecipitated complexes were then resolved by 8% SDS-PAGE and immunoblotted. Membranes were then probed with anti-NF-{kappa}B2 and anti-RelB Abs. Parallel immunoblotting of WCE with anti-GAPDH Abs was made to confirm equal loading.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we demonstrated that inhibition of the classical NF-{kappa}B activation pathway by the expression of a mutated I{kappa}B{alpha} resulted in DC incompetence to mature in response to TLR2, TLR3, and TLR4 engagement, although their ability to activate NF-{kappa}B undergoes phenotypic maturation upon T cell encounter and to prime alloreactive T cells both in vitro and in vivo remained intact.

Our data highlight once again the vast plasticity of DC (60). Even though p50/RelA dimers were previously reported to be necessary for the generation of CD11c+ DC in mice (61), we demonstrated that DC may adapt to the enforced inhibition of the classical NF-{kappa}B pathway by expanding the pool of the alternative pathway factors with increased expression of RelB, NF-{kappa}B2 p100, and IKK{alpha} protein levels. It is likely that NF-{kappa}B activity required for s-rI{kappa}B DC development and function occurs mainly through activation of the alternative pathway, which probably proceeds as a "salvage pathway." Indeed, a similar switch from the classical to the alternative pathway of NF-{kappa}B activation was recently reported in s-rI{kappa}B-expressing murine mammary gland cells in which p52/RelB dimers rescue an early delay in their development (57). The exact differentiation status of s-rI{kappa}B DC is nonetheless to be questioned. Despite growing slower than Ctrl DC, s-rI{kappa}B DC are neither apoptotic, nor necrotic at the end of the culture, but express very little CD11c, MHC II, and costimulatory molecules. However, all those markers are highly up-regulated at the surface of s-rI{kappa}B DC after allogeneic T cell contact, and the restoration of NF-{kappa}B activity allows them to elicit a classical DC phenotype. Our hypothesis is that s-rI{kappa}B DC are engaged to a certain extent in DC lineage but are inhibited in expressing a DC phenotype in the absence of T cell stimulation, which probably acts on s-rI{kappa}B DC as a final differentiation and maturation stimulus. One argument supporting this hypothesis is the normal expression of TLR2, TLR3, and TLR4 mRNA in s-rI{kappa}B DC, and the observation that s-rI{kappa}B DC encompass CCR5/CCR7-shifted mRNA synthesis upon LPS activation, allowing them to display normal migration properties. However, this last result was surprising because NF-{kappa}B is described as an important factor of CCR7 mRNA transcription (55, 56). Still, p38 MAPK and the transcription factor AP1 have also been implicated in CCR7 up-regulation in response to maturation stimuli (62, 63, 64, 65). Our data then indirectly show that the CCR5/CCR7 expression shift can occur in LPS-treated BM-DC, even in the absence of NF-{kappa}B activation, maybe by compensatory mechanisms.

The expression of a mutated I{kappa}B{alpha} in DC renders them defective to mature in response to several TLR ligands by inhibiting the classical NF-{kappa}B activation pathway and was associated with a lack of RelA nuclear translocation. More surprisingly, we also observed that LPS- or anti-LTbetaR-induced IKKbeta phosphorylation was totally undetectable in s-rI{kappa}B DC. This unexpected result requires further investigation to find an explanation. However, in our work, allogeneic s-rI{kappa}B DC still maintained their ability to undergo phenotypic maturation after prolonged contact with T cells. Moreover, we found that the maturation process in s-rI{kappa}B DC was preceded by the restoration of NF-{kappa}B-binding activity, which was further demonstrated to be mainly composed of p52 and RelB proteins. This strongly suggests that, although NF-{kappa}B activation through the classical pathway is broadly repressed in s-rI{kappa}B DC, these cells remain totally competent to activate NF-{kappa}B through the I{kappa}B-independent alternative pathway. Furthermore, we showed that activation of s-rI{kappa}B DC through LTbetaR, a well-known activator of the alternative pathway of NF-{kappa}B activation, triggers IKK{alpha} phosphorylation and nuclear translocation of p52/RelB dimers after NF-{kappa}B2 p100 processing, hereby reporting for the first time the alternative pathway of NF-{kappa}B activation in DC. Moreover, we observed that the activation of the alternative pathway of NF-{kappa}B in s-rI{kappa}B DC was not associated with activation of early classical NF-{kappa}B dimers (data not shown), as it was demonstrated for anti-LTbetaR-activated MEF (33, 59). Still, p100 processing was already demonstrated to occur independently of early NF-{kappa}B activation in 293 cells transfected with the EBV-derived protein LMP1 (66). The occurrence and dependency of early classical NF-{kappa}B activation events to achieve late NF-{kappa}B activation by the alternative pathway could then be variable, considering the type of cells or stimulus. In the case of DC generated from bone marrow progenitors, this variability could be further increased considering the length of DC generation, or the cytokines used to drive DC differentiation (with or without IL-4 added to GM-CSF). Nonetheless, our data are in contrast with the negative regulatory role of NF-{kappa}B2 p100 that has been described so far in DC. Indeed, NF-{kappa}B2-deficient mice were reported to exhibit enhanced RelB activity accompanied with a more mature phenotype and enhanced ability to prime CD4+ T cells (67). LTbetaR-induced nuclear translocation of p52/RelB dimers seems to occur in a different manner in Ctrl DC. In fact, we observed that NF-{kappa}B2 p100 appeared to be constitutively processed to p52 since it was undetectable in the cytoplasm of resting Ctrl DC. Additionally, we confirmed that cytoplasmic RelB, which is described to be mostly associated with NF-{kappa}B2 p100 in HeLa cells and MEF (33, 68), was completely absent in resting Ctrl DC. Although the emergence of nuclear p52 in LTbetaR-stimulated Ctrl DC was correlated with its cytoplasmic disappearance, the increase of nuclear RelB in anti-LTbetaR mAb-treated Ctrl DC is likely due to de novo protein synthesis, as it was previously reported for the LIGHT-induced generation of DNA-binding p52 in HeLa cells (34). The mechanism underlying LTbetaR-induced nuclear translocation of p52/RelB dimers in Ctrl DC needs further study.

The role of LTbetaR in T cell-driven s-rI{kappa}B DC maturation is still unknown. Although many reports already described LTbetaR as a potent inducer of the I{kappa}B-independent alternative pathway of NF-{kappa}B activation in MEF or HeLa cells (33, 34, 59), its expression on murine DC was mainly reported to be critical for their homeostasis and expansion (69, 70). Until now, the literature only described a possible role of LTbetaR in DC maturation since LT{alpha}–/–, LTbeta–/–, and LTbetaR–/– mice have reduced mature DC in the spleen (71), yet these observations were not linked to the activation of the alternative pathway of NF-{kappa}B activation in DC. Still, engagement of LTbetaR by activated T cells has already been demonstrated to induce cytokine release from bone marrow-derived mast cells (72). If s-rI{kappa}B DC maturation and antigenic presentation depends on LT{alpha}1beta2 or LIGHT expression on activated T cells, then how are naive allogeneic T cells initially activated? An explanation could be found in the report that murine naive CD4+ and CD8+ T cells express low but constitutive levels of LT{alpha}1beta2 (73). Since resting s-rI{kappa}B DC express LTbetaR, it is plausible that s-rI{kappa}B DC maturation starts with T cell encounter and then maturing s-rI{kappa}B DC amplify T cell activation by which both cell types reach maturation and full activation via positive feedback loops. Among the early cross-talks between DC and T cells, it is likely that up-regulation of CD40 occurs at the surface of s-rI{kappa}B DC. Indeed, CD40 receptor is also known to be a potent activator of the alternative pathway of NF-{kappa}B, and CD40/CD40L interactions have long been recognized as major activation signals at the DC-T cell interface (74). CD40/CD40L interaction could then be implicated in both differentiation/maturation of s-rI{kappa}B DC and in T cell-priming activity.

We also observed that allogeneic s-rI{kappa}B DC are able to prime T cell responses in vitro and in vivo, in contrast to previous data reporting on NF-{kappa}B ODN-treated or NF-{kappa}B-inhibiting drug-treated DC (12, 18). Our data are then in contrast with the familiar idea that DC immaturity induces tolerance, whereas mature DC are involved in immune priming. In fact, DC ability to induce tolerance or suppress primed immune responses has already been correlated with the absence of nuclear RelB (15), which further inhibits expression of CD40 molecules. Since RelB-containing NF-{kappa}B activity was detected in s-rI{kappa}B DC after in vitro T cell encounter or LTbetaR engagement, it is not surprising that s-rI{kappa}B DC are not tolerogenic. Targeted blocking of certain NF-{kappa}B subunits (in particular RelB) and inhibition of specific activation pathways engaged in DC by contact with T cells may then be more efficient than general DC immaturity to induce tolerogenic DC.

The physiological relevance of T cell-induced activation of the alternative NF-{kappa}B pathway in DC requires further investigation. Evidence from literature indicates that DC may need engagement of receptors triggering the alternative pathway of NF-{kappa}B activation to elicit certain types of immune responses. Indeed, DC stimulated through CD40 were shown to have a unique capacity to generate CD8+ CTL responses, whereas LPS-matured DC do not (75). These DC also display a different panel of cytokines (76) and encompass sustained NF-{kappa}B activation of RelB-containing dimers (77). Other studies have demonstrated that LTbetaR-deficient mice showed defective antiviral immunity (78) and that LT{alpha}1beta2 and LIGHT molecules are not only important for secondary lymphoid organ homeostasis but also play an important role in cellular immunity or in immune responses against limiting amounts of Ags (79, 80). Moreover, recent findings highlighted that some IKK{alpha} target genes, like organogenic chemokine genes, were identified to have specific {kappa}B binding sites for p52/RelB dimers (81). Taken together, these data strongly support the hypothesis that DC may induce various adaptive immune responses through activation of different NF-{kappa}B pathways, but also suggest that experimental protocols for the prevention of autoimmune diseases or allograft rejection through NF-{kappa}B impairment should consider modulation of these multiple activation pathways.


   Acknowledgments
 
We thank Claude Habran, Marie-Line Vanderhaeghen, Hans Schiffer, and Valerie Lebon for technical assistance; Olivier Vosters, Serge Vanden Eijnden, and Jolyn Johnson for sharing useful chemicals and Abs; Didier Blocklet and Serge Goldman from the Department of Nuclear Medicine and PET/Biomedical Cyclotron Unit, CUB-Hopital Erasme, Université Libre de Bruxelles, for help in DC migration assays; and animalists for mice caring. We also thank Fabrice Bureau for critically reading the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 The Institute for Medical Immunology is sponsored by the government of the Walloon Region and GlaxoSmithKline Biologicals. This study was also supported by the Fonds National de la Recherche Scientifique (FNRS, Belgium) and an Interuniversity Attraction Pole of the Belgian Federal Science Policy. F.M. is supported by a Formation à la Recherche dans l’Industrie et dans l’Agriculture grant from FNRS, S.B. is a scientific collaborator of the FNRS, S.G. is a postdoctoral fellow of FNRS, and V.F. is a research fellow of FNRS. Back

2 Address reprint requests to Dr. Véronique Flamand, Institute of Medical Immunology, 8 rue A. Bolland, B-6041 Gosselies, Belgium. E-mail address: vflamand{at}ulb.ac.be Back

3 Abbreviations used in this paper: DC, dendritic cell; BM-DC, bone marrow-derived DC; Ctrl DC, control-transduced DC; eGFP, enhanced GFP; IKK, I{kappa}B kinase; LN, lymph node; LTbetaR, lymphotoxin beta receptor; Pam3CSK4, S-[2.3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-Lys4-OH) trihydrochloride; poly(I:C), polyinosine-polycytidylic acid; s-rI{kappa}B, superrepressor of the classical NF-{kappa}B pathway; WCE, whole cell extract; MEF, mouse embryonic fibroblast; LIGHT, homologous to lymphotoxin, exhibits inducible expression, competes with herpesvirus glycoprotein D for HVEM on T cells. Back

Received for publication June 21, 2006. Accepted for publication November 9, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Hackstein, H., A. E. Morelli, A. W. Thomson. 2001. Designer dendritic cells for tolerance induction: guided not misguided missiles. Trends Immunol. 22: 437-442. [Medline]
  2. Roncarolo, M. G., M. K. Levings, C. Traversari. 2001. Differentiation of T regulatory cells by immature dendritic cells. J. Exp. Med. 193: F5-F9. [Free Full Text]
  3. Lu, L., A. W. Thomson. 2002. Manipulation of dendritic cells for tolerance induction in transplantation and autoimmune disease. Transplantation 73: S19-S22. [Medline]
  4. Morelli, A. E., A. W. Thomson. 2003. Dendritic cells: regulators of alloimmunity and opportunities for tolerance induction. Immunol. Rev. 196: 125-146. [Medline]
  5. Lutz, M. B., G. Schuler. 2002. Immature, semi-mature and fully mature dendritic cells: which signals induce tolerance or immunity?. Trends Immunol. 23: 445-449. [Medline]
  6. Mahnke, K., A. H. Enk. 2005. Dendritic cells: key cells for the induction of regulatory T cells?. Curr. Top. Microbiol. Immunol. 293: 133-150. [Medline]
  7. Steinman, R. M., D. Hawiger, M. C. Nussenzweig. 2003. Tolerogenic dendritic cells. Annu. Rev. Immunol. 21: 685-711. [Medline]
  8. Mahnke, K., E. Schmitt, L. Bonifaz, A. H. Enk, H. Jonuleit. 2002. Immature, but not inactive: the tolerogenic function of immature dendritic cells. Immunol. Cell Biol. 80: 477-483. [Medline]
  9. Tan, J. K., H. C. O’Neill. 2005. Maturation requirements for dendritic cells in T cell stimulation leading to tolerance versus immunity. J. Leukocyte Biol. 78: 319-324. [Abstract/Free Full Text]
  10. Giannoukakis, N., C. A. Bonham, S. Qian, Z. Chen, L. Peng, J. Harnaha, W. Li, A. W. Thomson, J. J. Fung, P. D. Robbins, L. Lu. 2000. Prolongation of cardiac allograft survival using dendritic cells treated with NF-{kappa}B decoy oligodeoxyribonucleotides. Mol. Ther. 1: 430-437. [Medline]
  11. Xu, M. Q., Y. P. Suo, J. P. Gong, M. M. Zhang, L. N. Yan. 2004. Prolongation of liver allograft survival by dendritic cells modified with NF-{kappa}B decoy oligodeoxynucleotides. World J. Gastroenterol. 10: 2361-2368. [Medline]
  12. Verhasselt, V., W. Vanden Berghe, N. Vanderheyde, F. Willems, G. Haegeman, M. Goldman. 1999. N-acetyl-L-cysteine inhibits primary human T cell responses at the dendritic cell level: association with NF-{kappa}B inhibition. J. Immunol. 162: 2569-2574. [Abstract/Free Full Text]
  13. Yoshimura, S., J. Bondeson, F. M. Brennan, B. M. Foxwell, M. Feldmann. 2001. Role of NF{kappa}B in antigen presentation and development of regulatory T cells elucidated by treatment of dendritic cells with the proteasome inhibitor PSI. Eur. J. Immunol. 31: 1883-1893. [Medline]
  14. Boffa, D. J., B. Feng, V. Sharma, R. Dematteo, G. Miller, M. Suthanthiran, R. Nunez, H. C. Liou. 2003. Selective loss of c-Rel compromises dendritic cell activation of T lymphocytes. Cell. Immunol. 222: 105-115. [Medline]
  15. Martin, E., B. O’Sullivan, P. Low, R. Thomas. 2003. Antigen-specific suppression of a primed immune response by dendritic cells mediated by regulatory T cells secreting interleukin-10. Immunity 18: 155-167. [Medline]
  16. Iruretagoyena, M. I., S. E. Sepulveda, J. P. Lezana, M. Hermoso, M. Bronfman, M. A. Gutierrez, S. H. Jacobelli, A. M. Kalergis. 2006. Inhibition of NF-{kappa}B enhances the capacity of immature dendritic cells to induce antigen-specific tolerance in experimental autoimmune encephalomyelitis. J. Pharmacol. Exp. Ther. 318: 59-67. [Abstract/Free Full Text]
  17. Ma, L., S. Qian, X. Liang, L. Wang, J. E. Woodward, N. Giannoukakis, P. D. Robbins, S. Bertera, M. Trucco, J. J. Fung, L. Lu. 2003. Prevention of diabetes in NOD mice by administration of dendritic cells deficient in nuclear transcription factor-{kappa}B activity. Diabetes 52: 1976-1985. [Abstract/Free Full Text]
  18. Tiao, M. M., L. Lu, R. Tao, L. Wang, J. J. Fung, S. Qian. 2005. Prolongation of cardiac allograft survival by systemic administration of immature recipient dendritic cells deficient in NF-{kappa}B activity. Ann. Surg. 241: 497-505. [Medline]
  19. Jimi, E., S. Ghosh. 2005. Role of nuclear factor {kappa}B in the immune system and bone. Immunol. Rev. 208: 80-87. [Medline]
  20. Karin, M., F. R. Greten. 2005. NF-{kappa}B: linking inflammation and immunity to cancer development and progression. Nat. Rev. Immunol. 5: 749-759. [Medline]
  21. Karin, M., A. Lin. 2002. NF-{kappa}B at the crossroads of life and death. Nat. Immunol. 3: 221-227. [Medline]
  22. Hayden, M. S., S. Ghosh. 2004. Signaling to NF-{kappa}B. Genes Dev. 18: 2195-2224. [Abstract/Free Full Text]
  23. Karin, M., Y. Ben Neriah. 2000. Phosphorylation meets ubiquitination: the control of NF-{kappa}B activity. Annu. Rev. Immunol. 18: 621-663. [Medline]
  24. Whiteside, S. T., A. Israel. 1997. I{kappa}B proteins: structure, function and regulation. Semin. Cancer Biol. 8: 75-82. [Medline]
  25. Chen, Z. J.. 2005. Ubiquitin signalling in the NF-{kappa}B pathway. Nat. Cell Biol. 7: 758-765. [Medline]
  26. Bonizzi, G., M. Karin. 2004. The two NF-{kappa}B activation pathways and their role in innate and adaptive immunity. Trends Immunol. 25: 280-288. [Medline]
  27. Senftleben, U., Y. Cao, G. Xiao, F. R. Greten, G. Krahn, G. Bonizzi, Y. Chen, Y. Hu, A. Fong, S. C. Sun, M. Karin. 2001. Activation by IKK{alpha} of a second, evolutionary conserved, NF-{kappa}B signaling pathway. Science 293: 1495-1499. [Abstract/Free Full Text]
  28. Weih, F., J. Caamano. 2003. Regulation of secondary lymphoid organ development by the nuclear factor-{kappa}B signal transduction pathway. Immunol. Rev. 195: 91-105. [Medline]
  29. Alcamo, E., N. Hacohen, L. C. Schulte, P. D. Rennert, R. O. Hynes, D. Baltimore. 2002. Requirement for the NF-{kappa}B family member RelA in the development of secondary lymphoid organs. J. Exp. Med. 195: 233-244. [Abstract/Free Full Text]
  30. Yilmaz, Z. B., D. S. Weih, V. Sivakumar, F. Weih. 2003. RelB is required for Peyer’s patch development: differential regulation of p52-RelB by lymphotoxin and TNF. EMBO J. 22: 121-130. [Medline]
  31. Tumanov, A. V., S. I. Grivennikov, A. N. Shakhov, S. A. Rybtsov, E. P. Koroleva, J. Takeda, S. A. Nedospasov, D. V. Kuprash. 2003. Dissecting the role of lymphotoxin in lymphoid organs by conditional targeting. Immunol. Rev. 195: 106-116. [Medline]
  32. Lo, J. C., S. Basak, E. S. James, R. S. Quiambo, M. C. Kinsella, M. L. Alegre, F. Weih, G. Franzoso, A. Hoffmann, Y. X. Fu. 2006. Coordination between NF-{kappa}B family members p50 and p52 is essential for mediating LTbetaR signals in the development and organization of secondary lymphoid tissues. Blood 107: 1048-1055. [Abstract/Free Full Text]
  33. Derudder, E., E. Dejardin, L. L. Pritchard, D. R. Green, M. Korner, V. Baud. 2003. RelB/p50 dimers are differentially regulated by tumor necrosis factor {alpha} and lymphotoxin-beta receptor activation: critical roles for p100. J. Biol. Chem. 278: 23278-23284. [Abstract/Free Full Text]
  34. Mordmuller, B., D. Krappmann, M. Esen, E. Wegener, C. Scheidereit. 2003. Lymphotoxin and lipopolysaccharide induce NF-{kappa}B-p52 generation by a co-translational mechanism. EMBO Rep. 4: 82-87. [Medline]
  35. Huang, D. B., D. Vu, G. Ghosh. 2005. NF-{kappa}B RelB forms an intertwined homodimer. Structure 13: 1365-1373. [Medline]
  36. Ryseck, R. P., F. Weih, D. Carrasco, R. Bravo. 1996. RelB, a member of the Rel/NF-{kappa}B family of transcription factors. Braz. J. Med. Biol. Res. 29: 895-903. [Medline]
  37. Burkly, L., C. Hession, L. Ogata, C. Reilly, L. A. Marconi, D. Olson, R. Tizard, R. Cate, D. Lo. 1995. Expression of relB is required for the development of thymic medulla and dendritic cells. Nature 373: 531-536. [Medline]
  38. Wu, L., A. D’Amico, K. D. Winkel, M. Suter, D. Lo, K. Shortman. 1998. RelB is essential for the development of myeloid-related CD8{alpha}– dendritic cells but not of lymphoid-related CD8{alpha}+ dendritic cells. Immunity 9: 839-847. [Medline]
  39. Zanetti, M., P. Castiglioni, S. Schoenberger, M. Gerloni. 2003. The role of relB in regulating the adaptive immune response. Ann. NY Acad. Sci. 987: 249-257. [Medline]
  40. Lernbecher, T., B. Kistler, T. Wirth. 1994. Two distinct mechanisms contribute to the constitutive activation of RelB in lymphoid cells. EMBO J. 13: 4060-4069. [Medline]
  41. Weih, F., S. A. Lira, R. Bravo. 1996. Overexpression of RelB in transgenic mice does not affect I{kappa}B{alpha} levels: differential regulation of RelA and RelB by the inhibitor protein. Oncogene 12: 445-449. [Medline]
  42. Murphy, K. M., A. B. Heimberger, D. Y. Loh. 1990. Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo. Science 250: 1720-1723. [Abstract/Free Full Text]
  43. Buonocore, S., F. Paulart, A. Le Moine, M. Braun, I. Salmon, S. Van Meirvenne, K. Thielemans, M. Goldman, V. Flamand. 2003. Dendritic cells overexpressing CD95 (Fas) ligand elicit vigorous allospecific T cell responses in vivo. Blood 101: 1469-1476. [Abstract/Free Full Text]
  44. Vermes, I., C. Haanen, H. Steffens-Nakken, C. Reutelingsperger. 1995. A novel assay for apoptosis: flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J. Immunol. Methods 184: 39-51. [Medline]
  45. Pear, W. S., G. P. Nolan, M. L. Scott, D. Baltimore. 1993. Production of high-titer helper-free retroviruses by transient transfection. Proc. Natl. Acad. Sci. USA 90: 8392-8396. [Abstract/Free Full Text]
  46. Osborn, L., S. Kunkel, G. J. Nabel. 1989. Tumor necrosis factor {alpha} and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor {kappa}B. Proc. Natl. Acad. Sci. USA 86: 2336-2340. [Abstract/Free Full Text]
  47. Duyao, M. P., A. J. Buckler, G. E. Sonenshein. 1990. Interaction of an NF-{kappa}B-like factor with a site upstream of the c-myc promoter. Proc. Natl. Acad. Sci. USA 87: 4727-4731. [Abstract/Free Full Text]
  48. Van Lint, C., C. A. Amella, S. Emiliani, M. John, T. Jie, E. Verdin. 1997. Transcription factor binding sites downstream of the human immunodeficiency virus type 1 transcription start site are important for virus infectivity. J. Virol. 71: 6113-6127. [Abstract]
  49. Yoshimura, S., J. Bondeson, B. M. Foxwell, F. M. Brennan, M. Feldmann. 2001. Effective antigen presentation by dendritic cells is NF-{kappa}B dependent: coordinate regulation of MHC, co-stimulatory molecules and cytokines. Int. Immunol. 13: 675-683. [Abstract/Free Full Text]
  50. Yamamoto, M., S. Sato, H. Hemmi, S. Uematsu, K. Hoshino, T. Kaisho, O. Takeuchi, K. Takeda, S. Akira. 2003. TRAM is specifically involved in the Toll-like receptor 4-mediated MyD88-independent signaling pathway. Nat. Immunol. 4: 1144-1150. [Medline]
  51. Akira, S., K. Takeda. 2004. Toll-like receptor signalling. Nat. Rev. Immunol. 4: 499-511. [Medline]
  52. Caux, C., S. Ait-Yahia, K. Chemin, O. de Bouteiller, M. C. Dieu-Nosjean, B. Homey, C. Massacrier, B. Vanbervliet, A. Zlotnik, A. Vicari. 2000. Dendritic cell biology and regulation of dendritic cell trafficking by chemokines. Springer Semin. Immunopathol. 22: 345-369. [Medline]
  53. Caux, C., B. Vanbervliet, C. Massacrier, S. Ait-Yahia, C. Vaure, K. Chemin, M. C. Dieu-Nosjean, A. Vicari. 2002. Regulation of dendritic cell recruitment by chemokines. Transplantation 73: (1 Suppl.):S7-S11. [Medline]
  54. Gunn, M. D.. 2003. Chemokine mediated control of dendritic cell migration and function. Semin. Immunol. 15: 271-276. [Medline]
  55. Krappmann, D., E. Wegener, Y. Sunami, M. Esen, A. Thiel, B. Mordmuller, C. Scheidereit. 2004. The I{kappa}B kinase complex and NF-{kappa}B act as master regulators of lipopolysaccharide-induced gene expression and control subordinate activation of AP-1. Mol. Cell. Biol. 24: 6488-6500. [Abstract/Free Full Text]
  56. Hinz, M., P. Lemke, I. Anagnostopoulos, C. Hacker, D. Krappmann, S. Mathas, B. Dorken, M. Zenke, H. Stein, C. Scheidereit. 2002. Nuclear factor {kappa}B-dependent gene expression profiling of Hodgkin’s disease tumor cells, pathogenetic significance, and link to constitutive signal transducer and activator of transcription 5a activity. J. Exp. Med. 196: 605-617. [Abstract/Free Full Text]
  57. Demicco, E. G., K. T. Kavanagh, R. Romieu-Mourez, X. Wang, S. R. Shin, E. Landesman-Bollag, D. C. Seldin, G. E. Sonenshein. 2005. RelB/p52 NF-{kappa}B complexes rescue an early delay in mammary gland development in transgenic mice with targeted superrepressor I{kappa}B-{alpha} expression and promote carcinogenesis of the mammary gland. Mol. Cell. Biol. 25: 10136-10147. [Abstract/Free Full Text]
  58. Browning, J. L., I. D. Sizing, P. Lawton, P. R. Bourdon, P. D. Rennert, G. R. Majeau, C. M. Ambrose, C. Hession, K. Miatkowski, D. A. Griffiths, et al 1997. Characterization of lymphotoxin-{alpha}beta complexes on the surface of mouse lymphocytes. J. Immunol. 159: 3288-3298. [Abstract]
  59. Muller, J. R., U. Siebenlist. 2003. Lymphotoxin beta receptor induces sequential activation of distinct NF-{kappa}B factors via separate signaling pathways. J. Biol. Chem. 278: 12006-12012. [Abstract/Free Full Text]
  60. Liu, Y. J., H. Kanzler, V. Soumelis, M. Gilliet. 2001. Dendritic cell lineage, plasticity and cross-regulation. Nat. Immunol. 2: 585-589. [Medline]
  61. Ouaaz, F., J. Arron, Y. Zheng, Y. Choi, A. A. Beg. 2002. Dendritic cell development and survival require distinct NF-{kappa}B subunits. Immunity 16: 257-270. [Medline]
  62. Ardeshna, K. M., A. R. Pizzey, S. J. Walker, S. Devereux, A. Khwaja. 2002. The upregulation of CC chemokine receptor 7 and the increased migration of maturing dendritic cells to macrophage inflammatory protein 3beta and secondary lymphoid chemokine is mediated by the p38 stress-activated protein kinase pathway. Br. J. Haematol. 119: 826-829. [Medline]
  63. Boisleve, F., S. Kerdine-Romer, M. Pallardy. 2005. Implication of the MAPK pathways in the maturation of human dendritic cells induced by nickel and TNF-{alpha}. Toxicology 206: 233-244. [Medline]
  64. Mathas, S., M. Hinz, I. Anagnostopoulos, D. Krappmann, A. Lietz, F. Jundt, K. Bommert, F. Mechta-Grigoriou, H. Stein, B. Dorken, C. Scheidereit. 2002. Aberrantly expressed c-Jun and JunB are a hallmark of Hodgkin lymphoma cells, stimulate proliferation and synergize with NF-{kappa}B. EMBO J. 21: 4104-4113. [Medline]
  65. Randolph, G. J., G. Sanchez-Schmitz, V. Angeli. 2005. Factors and signals that govern the migration of dendritic cells via lymphatics: recent advances. Springer Semin. Immunopathol. 26: 273-287. [Medline]
  66. Atkinson, P. G., H. J. Coope, M. Rowe, S. C. Ley. 2003. Latent membrane protein 1 of Epstein-Barr virus stimulates processing of NF-{kappa}B2 p100 to p52. J. Biol. Chem. 278: 51134-51142. [Abstract/Free Full Text]
  67. Speirs, K., L. Lieberman, J. Caamano, C. A. Hunter, P. Scott. 2004. Cutting edge: NF-{kappa}B2 is a negative regulator of dendritic cell function. J. Immunol. 172: 752-756. [Abstract/Free Full Text]
  68. Solan, N. J., H. Miyoshi, E. M. Carmona, G. D. Bren, C. V. Paya. 2002. RelB cellular regulation and transcriptional activity are regulated by p100. J. Biol. Chem. 277: 1405-1418. [Abstract/Free Full Text]
  69. Wang, Y. G., K. D. Kim, J. Wang, P. Yu, Y. X. Fu. 2005. Stimulating lymphotoxin beta receptor on the dendritic cells is critical for their homeostasis and expansion. J. Immunol. 175: 6997-7002. [Abstract/Free Full Text]
  70. Kabashima, K., T. A. Banks, K. M. Ansel, T. T. Lu, C. F. Ware, J. G. Cyster. 2005. Intrinsic lymphotoxin-beta receptor requirement for homeostasis of lymphoid tissue dendritic cells. Immunity 22: 439-450. [Medline]
  71. Abe, K., F. O. Yarovinsky, T. Murakami, A. N. Shakhov, A. V. Tumanov, D. Ito, L. N. Drutskaya, K. Pfeffer, D. V. Kuprash, K. L. Komschlies, S. A. Nedospasov. 2003. Distinct contributions of TNF and LT cytokines to the development of dendritic cells in vitro and their recruitment in vivo. Blood 101: 1477-1483. [Abstract/Free Full Text]
  72. Stopfer, P., D. N. Mannel, T. Hehlgans. 2004. Lymphotoxin-beta receptor activation by activated T cells induces cytokine release from mouse bone marrow-derived mast cells. J. Immunol. 172: 7459-7465. [Abstract/Free Full Text]
  73. Luther, S. A., A. Bidgol, D. C. Hargreaves, A. Schmidt, Y. Xu, J. Paniyadi, M. Matloubian, J. G. Cyster. 2002. Differing activities of homeostatic chemokines CCL19, CCL21, and CXCL12 in lymphocyte and dendritic cell recruitment and lymphoid neogenesis. J. Immunol. 169: 424-433. [Abstract/Free Full Text]
  74. Andreakos, E., C. Smith, C. Monaco, F. M. Brennan, B. M. Foxwell, M. Feldmann. 2003. I{kappa}B kinase 2 but not NF-{kappa}B-inducing kinase is essential for effective DC antigen presentation in the allogeneic mixed lymphocyte reaction. Blood 101: 983-991. [Abstract/Free Full Text]
  75. Kelleher, M., P. C. Beverley. 2001. Lipopolysaccharide modulation of dendritic cells is insufficient to mature dendritic cells to generate CTLs from naive polyclonal CD8+ T cells in vitro, whereas CD40 ligation is essential. J. Immunol. 167: 6247-6255. [Abstract/Free Full Text]
  76. Morelli, A. E., A. F. Zahorchak, A. T. Larregina, B. L. Colvin, A. J. Logar, T. Takayama, L. D. Falo, A. W. Thomson. 2001. Cytokine production by mouse myeloid dendritic cells in relation to differentiation and terminal maturation induced by lipopolysaccharide or CD40 ligation. Blood 98: 1512-1523. [Abstract/Free Full Text]
  77. O’Sullivan, B. J., R. Thomas. 2002. CD40 ligation conditions dendritic cell antigen-presenting function through sustained activation of NF-{kappa}B. J. Immunol. 168: 5491-5498. [Abstract/Free Full Text]
  78. Berger, D. P., D. Naniche, M. T. Crowley, P. A. Koni, R. A. Flavell, M. B. Oldstone. 1999. Lymphotoxin-beta-deficient mice show defective antiviral immunity. Virology 260: 136-147. [Medline]
  79. Granger, S. W., S. Rickert. 2003. LIGHT-HVEM signaling and the regulation of T cell-mediated immunity. Cytokine Growth Factor Rev. 14: 289-296. [Medline]
  80. Junt, T., A. V. Tumanov, N. Harris, M. Heikenwalder, N. Zeller, D. V. Kuprash, A. Aguzzi, B. Ludewig, S. A. Nedospasov, R. M. Zinkernagel. 2006. Expression of lymphotoxin beta governs immunity at two distinct levels. Eur. J. Immunol. 36: 2061-2075. [Medline]
  81. Bonizzi, G., M. Bebien, D. C. Otero, K. E. Johnson-Vroom, Y. Cao, D. Vu, A. G. Jegga, B. J. Aronow, G. Ghosh, R. C. Rickert, M. Karin. 2004. Activation of IKK{alpha} target genes depends on recognition of specific {kappa}B binding sites by RelB:p52 dimers. EMBO J. 23: 4202-4210. [Medline]



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